Following most forms of tissue injury, infection or inflammation, the concentrations of a number of plasma proteins increase, and then return to normal again as healing or recovery occurs. This process is known aas the acute phase response. In individuals with chronic inflammation, high levels of some acute phase proteins may persist.
Examples of acute phase reactants are C-reactive portein (CRP) and serum amyloid A protein (SAA). SAA is an .alpha..sub.1 globulin consisting of a single polypeptide chain of molecular weight between 11'500 and 14'000 Dalton. Its plasma concentration, which is normally around 1 .mu.g/ml or below in healthy individuals, increases substantially within few days following tissue injury or exposure to inflammatory stimuli. Related to the acute phase reactant CRP is serum amyloid P-component (SAP), a 9.5S .alpha..sub.1 glycoprotein of 235'000 Dalton molecular weight. Whereas mouse SAP concentration substantially increases in inflammation, human SAP is not a major acute phase reactant, although its level can be moderately increased in some chronic inflammatory diseases and malignancies [M. B. Pepys, Clinics in Immunology and Allergy, Vol. 1, p. 77-101 (1981)].
SAP and SAA are synthesized by hepatocytes after stimuli from activated macrophages. The SAA and SAP inducing factor has been shown to be similar if not identicl to the monokine interleukin-1.
SAA and SAP are immunologically identical with amuloid A fibrils and amyloid P-component, respectively, found as constituents of all amyloidosis deposits. Amyloidosis is a disease which occurs secondary to a chronic inflammation.
Changes in concentration and ratio of acute phase proteins, e.g. CRP and SAA, and of SAP are important for diagnosis and management purposes of a number of acute and chronic inflammatory diseases such as rheumatic conditions, e.g. rheumatoid arthritis, juvenile polyarthritis, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis or rheumatic fever, vasculitis syndromes, Chron's disease, autoimmune conditions, e.g. systemic lupus erythematosus or polymyositis, malignancies, transplant rejection and the like.
The usual test for measuring changes in actue phase and related proteins until recently has been the erythrocyte sedimentation rate. This test is cheap and easily performed, but as an indirect method, not very accurate and reproducible. With the development of antisera directed against these proteins, it is now possible to measure individual components of the acute phase response and gain valuable information for diagnostic purposes. CRP has been and is still the acute phase reactant most widely measured. But recent data suggest that SAA is a more sensitive marker of inflammation than CRP [R. E. Chambers et al., Annals of the Rheumatic Diseases, 42, 665 (1983)]. It is also becoming evident that not all acute phase proteins are raised in parallel and that further valuable information can be obtained from the assessment of the SAP level in plasma.
The known techniques for SAA quantification basically are radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) [G. Marhaug, Scand. J. Immunol. 18, 329 (1983)]. These assays in their present form are not very sensitive and lack high reproducibility. There is a need for a more sensitive and reliable assay which allows the determination of SAA in a minimal amount of serum with simple and safe reagents in the whole concentration range of 0.1 .mu.g/ml up to 2000 .mu.g/ml.
SAP usually is measured by rocket immunoelectrophoresis, by indirect competitive ELISA (requiring large amounts of purified SAP) or by competitive RIA (requiring the preparation and handling of radioactive material). All these techniques are not sufficiently sensitive and require large amounts of serum hampering routine analysis. A sensitive method is therefore required which allows the measurement of SAP in a small amount of serum in a safe and reproducible manner in a concentration range of 1 .mu.g/ml to 100 .mu.g/ml in serum.